Measuring Actin Flow in 3D Cell Protrusions Chi-Li Chiu, Michelle A. Digman, Enrico Gratton  Biophysical Journal  Volume 105, Issue 8, Pages 1746-1755 (October 2013) DOI: 10.1016/j.bpj.2013.07.057 Copyright © 2013 The Authors Terms and Conditions

Figure 1 Experimental design. (A) A four-leaf clover-shaped laser orbit (yellow) across a cell protrusion. 128 pixels are collected along the orbit, starting at the arrow. (B) The data are shown as a 2D pseudoimage where x axis represents the pixel number along the orbit and the y axis is time. Using this method, structures within the protrusion (blue) and at the periphery (green) can be detected. The cell boundary is defined by the interface of black (the background) and green. (C) Four orbits are collected sequentially with no time delay between them. Orbit 1, 2 and orbit 3, 4 are done at the same position, respectively. The distance between orbit 1, 2 and orbit 3, 4 is fixed. The orbit center is adjusted by nSPIRO feedback to be at the center of the protrusion. The four-orbit pattern is continuously repeated for several minutes of acquisition. (D) The molecule dynamics between pixels can be discerned using correlation functions. Not only the diffusion can be detected by correlating points along one orbit, but also the directional flow can be measured by correlating points at two adjacent orbits. The peak of the correlation function indicates the time a fluorescent structure takes to travel from one point to another. (E) If the cell protrusion contains an object indicated in red, for example an F-actin bundle, the pseudoimage will show the object at a specific location along the orbit as shown in (F). To see this figure in color, go online. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Fourth harmonic modulation of orbit intensity profile correlates with cylinder radius. (A) For a given radius of the four-clover patterned orbit (0.55 μm), a larger fluorescent cylinder radius results in smaller modulation amplitude. The modulation change is most significant at the fourth harmonic. (B) The relationship between the modulation amplitude at the fourth harmonic and the cylinder radius is largely independent on the noise (in the range 0–20%), especially at larger cylinder radius. The horizontal dotted line shows that the measured modulation of 0.17 corresponds to a cylinder radius of 1.12 μm. (C) Raster scan of a cell protrusion (top) and its cross-section scan through the dashed line (bottom). By performing an orbital scan using the clover pattern at a position along the cell protrusion we can obtain the amplitude of the fourth harmonic modulation and estimate the diameter of the cell protrusion at that location, as indicated by the circle at the bottom panel. (D) Determination of the position of coherent motion by the pair-correlation method in a protrusion that shows motion. The protrusion for this part D of the figure was larger (1.25 ± 0.01 μm) than the protrusion shown in D. The gray scale along the clover shape trajectory is based on the intensity profile, and the black dots (indicated by arrows) are the positions of detected actin flow by the pCF method. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Raster scan images of stable cell protrusion and protrusion tip of MDA-MB-231 cells in 3D type I collagen matrix. (A) Reflection signal from collagen (red) and actin-eGFP signal (green) were collected simultaneously. The red box indicates the protrusion chosen for the orbital scanning. (B) Orbital scan was performed on the cell protrusion at the red bar locations. The orbit radius was 1.46 μm and the distance between orbits was 0.5 μm. (C) MDA-MB-231 cell with active cell protrusion tip. (D) A smaller radius (1 μm) was used for orbital tracking whereas the distance between the orbits remained at 0.5 μm. To see this figure in color, go online. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Pair-correlation analysis of a stable protrusion. (A) The intensity pseudoimages of actin-eGFP at a stable protrusion at orbit 1 and orbit 2 (same position). (B) Pair-correlation of orbit 1 to orbit 2 (G12) and orbit 2 to orbit 1 (G21). Both show long correlation at specific positions, indicated by arrows. (C) The intensity pseudoimages of actin-eGFP at a stable protrusion at orbit 1 and orbit 3 (0.5 μm apart). (D) Pair-correlation of orbit 1 to orbit 3 (G13) and orbit 3 to orbit 1 (G31). No significant correlation can be seen from either direction. (E) The intensity pseudoimages of collagen reflection signal at orbit 1 and orbit 2 (same position). (F) Pair-correlation of orbit 1 to orbit 2 (G12) and orbit 2 to orbit 1 (G21). Both show long correlation at specific positions that are distinct from the actin-eGFP channel. (G and H) The intensity pseudoimages of collagen reflection signal at orbit 1 and orbit 3 (0.5 μm apart), and their corresponding correlation results. The long consistent correlation pattern suggests the stable microenvironment. To see this figure in color, go online. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 5 Pair-correlation analysis of an active protrusion tip. (A) The intensity pseudoimage of actin-eGFP at the protrusion tip at orbit 1 and orbit 2 (same position). (B) Pair-correlation of orbit 1 to orbit 2 (G12) and orbit 2 to orbit 1 (G21). Similar to the analysis from a stable protrusion, both correlation functions show long correlation at specific positions, indicated by arrows. (C and D) However, when correlating the intensity of actin-eGFP at the protrusion tip at orbit 1 and orbit 3 (0.5 μm apart), the flow (and its direction) was captured, as can be seen from the prominent peak in G13 (pointed by arrows). At the same location, G31 shows no significant correlation in the opposite direction. (E–H) For the collagen channel, the correlation at orbits at the same position shows long, consistent correlation, whereas the correlation of orbits 0.5 μm apart shows the pattern of flow similar to actin-eGFP nearby (panel H), suggesting the actin flow also changes the collagen position around the protrusion. To see this figure in color, go online. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 6 F-actin flow rate versus distance to the cell protrusion tip. (A) Two examples of actin flow profile along cell protrusions in 3D. In these two examples, the flow rate is slower close to the cell body. (B) Distance to protrusion tip versus actin flow rate for 37 independent measurements. The dashed line represents the detection limit. (C) With blebbistatin treatment, the percentage of measured actin flow with flow rate higher than 0.5 μm drops significantly compared to nontreated cells (P value < 0.01). Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions

Figure 7 F-actin position in 3D verified by Lifeact and pharmacological treatment. (A) Actin-eGFP (green) showed relatively homogeneous distribution in a cell except low intensity at the nucleus. Collagen SHG is shown in yellow. (B) Lifeact-eGFP showed much higher intensity near the cell periphery, in line with our observation of F-actin position using the correlation approach. (C) Pair-correlation of orbit 1 to orbit 2 (G12) of MDA-MB-231 without treatment (control), treated with latrunculin B, and treated with jasplakinolide. The red arrows indicate the position of long correlations corresponding to F-actin, which was abolished by the treatment with latrunculin B. Jasplakinolide, on the other hand, increased the long time correlations, indicating more stable F-actin. To see this figure in color, go online. Biophysical Journal 2013 105, 1746-1755DOI: (10.1016/j.bpj.2013.07.057) Copyright © 2013 The Authors Terms and Conditions